Janus porous polylactic acid membranes with versatile metal–phenolic interface for biomimetic periodontal bone regeneration

Conventional treatment to periodontal and many other bone defects requires the use of barrier membranes to guided tissue regeneration (GTR) and guided bone regeneration (GBR). However, current barrier membranes normally lack of the ability to actively regulate the bone repairing process. Herein, we proposed a biomimetic bone tissue engineering strategy enabled by a new type of Janus porous polylactic acid membrane (PLAM), which was fabricated by combining unidirectional evaporation-induced pore formation with subsequent self-assembly of a bioactive metal–phenolic network (MPN) nanointerface. The prepared PLAM-MPN simultaneously possesses barrier function on the dense side and bone-forming function on the porous side. In vitro, the presence of MPN nanointerface potently alleviated the proinflammatory polarization of mice bone marrow-derived macrophages (BMDMs), induced angiogenesis of human umbilical vein endothelial cells (HUVECs), and enhanced the attachment, migration and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). The implantation of PLAM-MPN into rat periodontal bone defects remarkably enhanced bone regeneration. This bioactive MPN nanointerface within a Janus porous membrane possesses versatile capacities to regulate cell physiology favoring bone regeneration, demonstrating great potential as GTR and GBR membranes for clinical applications.


Introduction
Periodontal bone defect caused by periodontitis, tumor or trauma can damage the integrity of periodontal tissue, affecting the aesthetics, occlusal function and quality of life of patients. The repair of theses defects and the regeneration of bone still remain major clinical challenges 1 . In recent years, guided tissue engineering (GTR) and guided bone regeneration (GBR) have emerged as important strategies to address these challenges, both of which involve the restoration of periodontium/bone defects by mean of barrier membranes 2,3 . Bone substitute materials are commonly used in combination with the barrier membrane during surgical implementation as the traditional barrier membrane does not have enough capability for bone regeneration, which is both time-consuming and costly for clinical practice [4][5][6] .
The natural biological process of bone regeneration includes immune cell response during the initial in ammatory stage, followed by endothelial vascular neogenesis and subsequent stem cell recruitment and osteogenic differentiation during the remodeling stage 7 . Therefore, an ideal GTR/GBR membrane is expected to possess superior capacities to actively regulate these bone-repair-related cells (e.g. immune cells, endothelial cells and stem cells), while avoiding faster-growing non-osteogenic brous cells or tissues from migrating into the defect to interfere with the bone regeneration process.
To realize these goals, numerous membranes with a bi-layered or "Janus" structure have been developed, including electrospun nano ber double-layer membranes, layer-by-layer assembled polymer/ceramic composites, and 3D-printed scaffolds with gradient porosities 8- 11 . In these studies, low-cost and easyprocessed polymers such as polylactic acid (PLA) have been widely used 8 . Nevertheless, since synthetic polymer substrates are normally lacking of bioactive sides to interact with bone-repair-related cells 12,13 , a simple bioactive polymer membrane with anisotropic structures for bone repair is highly demanded.
Assembly of nanostructured interfaces on biomaterials is proven to be an effective way for manipulating cell behavior to facilitate tissue regeneration 14 . Metal phenolic network (MPN), a complex supramolecular structure formed by the coordination of metal ions and phenolic ligands, has been widely studied as bioactive nanocoating for implant modi cation, drug release and stem cell differentiation [15][16][17] . It is reported that numerous naturally-derived phenolic molecules (e.g. tannic acid (TA) and catechins) are bene cial for anti-in ammatory and bone forming purposes 18, 19 . Metal ions such as copper ions (Cu 2+ ) have been considered to stimulate angiogenesis 20,21 . In addition, MPN is biodegradable in physiological environment 22 . Therefore, we hypothesized that introducing MPN nanocoating to a Janus porous membrane could provide a bioactive interface for immunomodulation, angiogenesis, stem cell regulation, that may achieve a biomimetic bone regeneration.
Herein, we reported a Janus porous PLA membrane (PLAM) modi ed with MPN nanointerface (PLAM-MPN) that was fabricated by combining unidirectional evaporation-induced pore formation with a subsequent interfacial self-assembly of TA and Cu 2+ . We envisioned that the acquired PLAM-MPN could possess barrier function on the dense side and bone-forming function on the MPN-coated porous side (Scheme 1). To test the hypothesis, we examined the ability of PLAM with different MPN nanointerface coatings to regulate bone-repair-related cells by studying their interactions with rat bone marrow-derived macrophages (BMDMs), human umbilical vein endothelial cells (HUVECs), and periodontal ligament stem cells (PDLSCs) in vitro. Furthermore, implantation of PLAM-MPN into a rat periodontal defect model was performed to investigate their potential for the biomimetic periodontal bone regeneration.

Experimental Section
2.1. Fabrication of the scaffolds PLAM were prepared by evaporative phase separation method. The preparation process was as follows: dichloromethane (DCM) was added to a glass bottle, and then polyethylene glycol (PEG) 200 was added to DCM, the mass ratio of PEG and DCM was 1:5. The mixture was vigorously stirred, then PLA (13.3% w/w DCM) was dispersed in the PEG solution, and the glass vial was sealed and placed under sonication at 37 ℃ to minimize solvent evaporation. The sonication was supplemented with stirring until the mixture was homogeneous. The mixture was poured over a glass dish to distribute evenly and the glass dish was then placed in a fume hood at room temperature for 24 h to remove residual solvent. Finally, by removing the thin lm (300-500 µm in thickness) from the glass plate, the Janus porous barrier lm was obtained.

Fabrication of the MPN
MPN was obtained by a layer-by-layer method. The preparation process was as follows: the prepared PLAM scaffold in the plate was washed three times with deionized water. Next, the PLAM scaffold (100 mm in diameter) was soaked with 10 mL of TA solution (0.8 mg mL −1 ) at room temperature, shaken for 30 s at 75 rpm, followed by the addition of 10 mL of Cu 2+ solution (0.2 mg mL −1 ), shaken again for 30 s at 75 rpm. Then, 100 μL of sodium hydroxide solution (pH=13) was added to adjust the pH to 7.5 and shaken for 30 s at 75 rpm. Finally, the scaffold was washed thrice with deionized water to obtain the MPN-loaded PLAM scaffold. According to the number of MPN layers required subsequently, the above preparation process was repeated 1, 5 and 10 times to obtain PLAM-1MPN (PLAM-1), PLAM-5MPN (PLAM-5) and PLAM-10MPN (PLAM-10) scaffolds, respectively.

Characterization of the scaffolds
The morphology of the scaffolds was observed using a scanning electron microscope (SEM) (Pro G5, Phenome) at an accelerating voltage of 5 kV. To corroborate the different chemical composition of the PLAM and the PLAM-MPN scaffolds, ultraviolet spectrophotometer (UV) (Shimadzu, UV2600 Ι) was used to detect the characteristic peak of Cu-TA. To detect whether Cu 2+ was loaded successfully on the surface of the PLAM-MPN scaffolds, Fourier infrared spectrometer (FTIR) (Bruker, ALPHA ) was used to scan over the range of 50-3500 cm −1 .
The surface wettability of the scaffolds was detected by static water contact angle measurement at room temperature (26±1 ℃). A contact angle instrument (Zhongchen, JC2000) was utilized, where 2.0 µL deionized water was automatically dropped onto the scaffold and recorded using a digital camera until the water droplet on the scaffold displayed a stable shape. Three points at least from different positions of each sample were recorded and averaged to determine the static water contact angle.
The tensile strength of scaffolds (10 × 50 mm) was characterized using a universal materials tester (Shimadzu, Japan). All samples were stretched at a constant tensile rate of 10 mm min −1 and the original length of the strips was 30 mm. Each sample was measured for three times in the tensile tests.

Cell culture
The following study protocol was approved by the Medical Ethical Committee of School of Stomatology, Shandong University (Protocol Number: GD20210609). The isolation of BMDMs was carried out based on previous procedures 23 .The BMDMs were cultured in DMEM medium contained 20% fetal bovine serum (FBS) (BioInd, Kibbutz, Israel) and 1% penicillin/streptomycin and 20 ng mL −1 of macrophage colony-stimulating factor (M-CSF) (Protein-tech, Chicago, USA). Fresh medium was changed regularly until 80-90% con uent monolayers were obtained.
The following study protocol was approved by the Medical Ethical Committee of School of Stomatology, Shandong University (Protocol Number: GR20210323). The isolation of human PDLSCs was carried out based on our previously reported procedures 24 . Brie y, human PDLSCs were cultured in DMEM supplemented with 10% FBS. PDLSCs at passages 4-6 were used for the following experiments. HUVECs were commercially purchased (ScienCell, San Diego, USA) and seeded in supplemented endothelial culture medium (ScienCell). All cells were cultured at 37 ℃ in a 5% CO 2 incubator with a humidi ed atmosphere.
The scaffolds were cut into 24 mm in diameter for 6 -well culture plate or 14 mm for 24 -well plate. The scaffold membranes were soaked in 75% ethanol for 2 h, after that these scaffolds were rinsed with PBS solution to remove residual ethanol. Then, the scaffolds were dried under sterile conditions and soaked in α-MEM for 24 h. BMDMs, PDLSCs and HUVECs were seeded on top of the scaffolds respectively for subsequent experiments.

Cell viability evaluation
The cytotoxicity of the PLAM-MPN and pure PLAM scaffolds was examined by a LIVE/DEAD viability/cytotoxicity kit (Invitrogen, CA, USA). PDLSCs were seeded at a density of 5×10 4 cells/well and incubated for 1, 2 and 3 d. At the end of each time period, the medium was discarded. The scaffolds were rinsed gently thrice with PBS. Next, the staining working solution were prepared according to the instructions: 5 μL calcein-am solution and 10 μL propidium iodide (PI) solution were respectively added to 5 mL PBS. Finally, the different samples were observed under the microscope and photographed. Cell viability and proliferation on different substrates were evaluated by CCK-8 (Dojindo Laboratories, Tokyo, Japan) after cultivated for 1, 2 and 3 d.

PDLSC morphology on the scaffolds
First, the growth state of PDLSCs on the scaffolds was observed by confocal laser scanning microscopy (CLSM, LSM880). In order to identify the porous structure of the scaffold, the red rhodamine B uorescent dye was added to the PLAM-B, PLAM-P, PLAM-1, PLAM-5, PLAM-10 surface. All the following experimental operations were performed in the dark. After 48 h culture, the medium was discarded. The scaffolds were rinsed gently thrice with PBS. Then, cells were xed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, the cells were stained with DAPI for 5 min. Finally, after washing again with PBS, the growth state and distribution of cells in the stained porous scaffolds were observed under CLSM. The morphology of PDLSCs on PLAM-10 scaffold was characterized under SEM after dehydration by gradient alcohol.

In vitro immunoregulation assay
The identity of the obtained BMDMs was tested rst by ow cytometry (Accuri-C6, BD Biosciences, San Diego, USA). The detected maker included the F4/80 (17-5920-82, Invitrogen) and CD11b (141708, Biolegend). After being passaged, the BMDMs were seeded onto the barrier membrane at a density of 2×10 5 cells/well. After culturing for 12 h, the conditioned medium was replaced. Among them, in all groups to detect the M1 polarization, the medium was DMEM containing 100 ng mL -1 LPS + 20 ng mL -1 IFN-γ in 10% FBS, and the stimulation time was 12 h. The groups to detect the M2 polarization, except that the medium of the "NC+IL-4" group was 20 ng mL -1 IL-4+20 ng mL -1 IL-10 in DMEM with 10% FBS, the rest medium in the group was DMEM with 10% FBS alone, and the stimulation time was 48 h. Next, phenotypic characterization of BMDMs induced by the different scaffolds was performed by ow cytometry. Stimulated BMDMs were incubated with allophycocyanin (APC)-conjugated anti-iNOS (2102823, Invitrogen) and anti-CD206 (MMR) (141707, Biolegend).
The gene expression levels of tumor necrosis factor (TNF-), inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1), and CD206 were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) to assess the immunoregulation effects of the PLAM-MPN scaffolds on BMDMs, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer was selected as the housekeeping gene and the sequences of the primers were provided in Supplementary Table S1.

In vitro vascularization assay
After the HUVECs were cultured with different scaffolds respectively for 14 d, the tubule formation assay was performed according to the manufacturer's instructions. The Matrigel were placed at 4 ℃ overnight and thaw and the 48-well culture plate and pipette tips were pre-cooled at -20 ℃ for 1 h in advance. Then, the thawed Matrigel was spread evenly on the pre-cooled 48-well plate (100 μL/well), placed at 37 ℃ for 30 min to solidify the Matrigel. While waiting for Matrigel to solidify, cells grown on negative control (NC), PLAM, and PLAM-MPN scaffolds were collected by trypsinization and washed twice with PBS. The cells were then resuspended at a density of 5.5×10 5 mL −1 . Finally, 100 μL of cell suspension was added to each well of a 48-well plate. After 8 h of incubation, the samples were observed and photographed under microscope. The relative genes (vascular endothelial growth factor (VEGF), stem cell factor (SCF) and hypoxia-inducible factor (HIF)) expression levels of angiogenic differentiation were analyzed by qRT-PCR. GAPDH primer was selected as the housekeeping gene and the sequences of the primers were provided in Supplementary Table S1.

In vitro stem cell recruitment assay
The PDLSCs were digested and adjusted to 5×10 5 mL −1 in α-MEM supplemented with 0.1% FBS, and 200 μL of the cell suspension were inoculated to the upper side of the migration chamber. The scaffolds were then placed in the lower side and soaked in 500 μL of α-MEM containing 0.1% FBS. The NC group was also cultured in α-MEM with 0.1% FBS, and the PC group was cultured with α-MEM with 10% FBS. After the cells incubated at 37 ℃ for 20 h, the noninvasive cells on the upper side were removed by a cotton swab. Finally, the cells were xed, stained with crystal violet and observed under an optical microscope (Olympus, Tokyo, Japan).

In vitro PDLSC osteogenesis assay
The PDLSCs were seeded at a density of 1.5×10 5 cells/well. After the cell adhesion, the medium was replaced with osteogenic induction medium. After 21 d, the samples were rinsed gently with PBS to avoid wrapping the cells on the culture plate. Then the cells were xed with 4% paraformaldehyde for 30 min. After being rinsed, an appropriate amount of Alizarin Red staining solution was added to each well for 30 min. A digital camera was used to collect the optical image of the overall macroscopic effect of the ori ce plate, and the microscopic image of the mineralized nodules was observed under a microscope and photographed. Next, 1 mL of 10% cetylpyridinium chloride (CPC) solution was added to each well of the 6-well plate. After the mineralized nodules were dissolved for 30 min at room temperature, 100 μL of the solution were pipetted into a 96-well plate. Finally, the OD value was measured and analyzed at 562 nm. The relative genes (alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteopontin (OPN)) expression levels of osteogenic differentiation were analyzed by qRT-PCR. GAPDH primer was selected as the housekeeping gene and the sequences of the primers were provided in Supplementary Table S1.

In vivo periodontal bone regeneration assay
Animal studies were approved by the Ethics Committee of Stomatological Hospital of Shandong University (Protocol Number: GD20190901). The animal experimental protocols were performed according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Wistar rats (male, 7 w, 220±20 g, Chales River, Beijing, China) were used in this study (24 rats for each group, 72 rats in total). they were stored in the animal center at the Shandong University following a 12+12 hours dark/light cycle with a fasten 20-25°C ambient temperature, food (provided and water were autoclavesterilized prior to administration. The mice were free to access to food and water prior and after experiments. According to the previous published articles 24,25 , the operation was performed under pentobarbital sodium anesthesia (40 mg kg −1 body weight). In brief, bilateral periodontal bone defects (5×4×1 mm 3 , L×H×D) were created with a dental drill, and the defect area was located approximately 1 mm posterior to the anterior border of the mandible and 1 mm below the superior border of the mandible. All rats with defects were numbered and randomly divided into 3 groups, in which different scaffolds were implanted into the defects: (1) no treatment (NC), (2) PLAM, and (3) PLAM-10. After surgery, the animals were intramuscularly injected with penicillin (400000 U mL −1 ) for three successive days. At 1, 2, 4 and 8 w, the rats were sacri ced by excessive pentobarbital anesthesia, then the mandible specimens of the rats were collected and xed with 4% paraformaldehyde by cardiac perfusion for the following experiments.

Micro-computed tomography assay
To estimate and compare bone regeneration of defect site in each group, micro-computed tomography (Micro-CT) (PerkinElmer, MA, USA) analysis was applied. Quantitatively, the parameters including the bone volume/total volume (BV/TV),bone surface/total volume (BS/TV), trabecular bone thickness (Tb.Th), trabecular bone separation (Tb.Sp) were analyzed and calculated to assess bone regeneration. The implanted scaffolds were taken out at 8 w post-surgery, and the cell growth on the both surfaces of the barrier membrane was observed by SEM.

Immuno uorescence assay
Decalci cation of the specimens was performed with 10% disodium ethylenediaminetetraacetic acid (EDTA-Na 2 , Solarbio Beijing, China) at 4 ℃ for 30 d. Then, decalci ed tissue embedded into para n wax was sliced into 5 μm sections. Immuno uorescent staining was performed as described before. In order to detect the bone regeneration, phenotype switching of macrophages and angiogenesis in vivo, the tissue sections were incubated with primary antibodies anti-ALP (1:500, ab65834, Abcam), anti-Runx2  300, ab28364, Abcam). The uorescent slides were nally covered by the cover glass using mounting medium containing DAPI. The uorescent images were obtained with the uorescence microscope, the uorescence intensity of ALP and the number of Runx2 + cells, CD206 + /CD68 + cells, iNOS + /CD68 + cells and CD31 + cells in the defects were counted and measured by Image J software.

Statistical analysis
All values are reported as the mean ± standard deviation (SD) and were analyzed by Student's t test and one-way ANOVA using GraphPad Prism software (Version 6, MacKiev Software, Boston, MA, USA). Differences were considered statistically signi cant at P < 0.05.

Characterization of the Janus porous membrane
The manufacturing process of the PLAM scaffold with the assembled MPN nanointerface is shown in Figure 1a. The morphology of the PLAM was characterized by SEM (Figure 1b), one side of the PLAM possessed a porous structure (denoted as PLAM-P), while the other side of the membrane showed a dense barrier structure (denoted as PLAM-B). As shown in Figure 1c-d, the pore size and porosity of the pristine PLAM could be adjusted by the amount of added PEG 26 . As PEG content was increased, the pore size of PLAM-P or PLAM-B side increased correspondingly, reaching the maximum value at 20% PEG. However, the ratio between the pore sizes in the two sides of the membrane decreased when the PEG content was higher than 10%, in favor of bigger pores in PLAM-B part. Considering that the pore size and porosity of the PLAM-P side is expected to be large enough to be conducive to the ingrowth of bone cells and that the PLAM-B side needs to be dense with small pore size and less porosity to act as a barrier, pristine PLAM prepared at 15% PEG with larger pore size was nally chosen for subsequent experiments.
Before harvesting the Janus porous PLAM from the drying plate, the pristine membranes were incubated with Cu 2+ and TA solution to initiate the MPN nano lm assembly on the porous side to obtain the nal PLAM-MPN with 1, 5, 10 MPN layers (PLAM-1, 5, 10). Figure 1e shows the UV-Vis absorption spectra of PLAM-MPN scaffolds, where the strong peak at 260-340 nm was attributed to the absorption caused by the chemical coordination of Cu-TA 27 . The FTIR spectra of PLAM-MPN (Figure 1f) evidenced the strong peak at 1759 cm −1 attributed to the absorption of the ester group and the absorption peaks at 1690-1500 cm −1 attributed to the C=C bending vibration of TA, respectively 28 .
The surface wettability of a scaffold is an important parameter affecting the overall performance of cell growth in tissue engineering 29 . The wettability of the scaffolds was evaluated by water contact angle (WCA) measurements (Figure 1g), the contact angle of the PLAM-10 scaffolds signi cantly reduced to 56.4°, while the contact angle of PLAM-B was as high as 89.0°. The anisotropic difference of hydrophilicity in PLAM could favor its GTR function by affecting cell attachment. Moreover, the bulk elastic modulus of the PLAM-MPN scaffolds signi cantly increased compared to that of PLAM ( Figure  1h). The mechanical enhancement could ensure that the scaffolds provide a su cient mechanical support for bone tissue regeneration.
The above results indicated the successful assembly of MPN nanointerface on the Janus porous PLAMs, endowing the PLAM-MPN scaffolds with anisotropic surface property and enhanced mechanical properties.

Stem cell viability, recruitment and attachment on the MPN nanointerface
To investigate the biocompatibility of the MPN nanointerface, PDLSCs were cultured onto PLAM-B, PLAM-P and PLAM-MPN formed with 1, 5, and 10 MPN layers. Live/dead staining results showed that there was no statistical difference among all groups except that PLAM-P increased the number of PDLSCs adhered to the scaffolds, indicating that the porous side of PLAM facilitated stem cell adhesion and proliferation, and the dense barrier side was not conducive to the attachment and growth of the cells and could be utilized as a barrier membrane (Figure 2a, b). Meanwhile, CCK-8 assay results were consistent with the Live/Dead staining (Figure 2c). The cell viability on each sample increased with prolonged culture time from 1 to 3 d, indicating that prepared membranes facilitated stem cell proliferation.
In addition, CLSM was used to observe the 3D attachment of PDLSCs on each sample. Except for PLAM-B group, PDLSCs well adhered onto the PLAM-P side of the scaffolds. Moreover, the cell penetration deepened with the increased thickness of MPN (Figure 2d, e), because the MPN coating is likely favorable for cell migration 16,30 . The detailed cell morphologies on the PLAM-10 could be clearly observed by SEM imaging, showing that the stem cell grew inside the pores and the cell body was well spread on the pore surface (Figure 2f).
The enhanced cell migration capacity of PDLSCs in the presence of the scaffolds was then investigated by performing the transwell migration experiment. As shown in Figure 2g and h, compared with the NC and PLAM scaffolds, the PLAM-10 scaffold signi cantly increased the number of migrated cells. The results indicate that MPN coating could stimulate cell migration in vitro, which is possibly due to the characteristics that TA-Cu MPN nanocoating is biodegradable and the release of Cu 2+ can mediate the migration of PDLSCs through the activated hypoxia inducible factor-1α (HIF-1α) pathway and the upregulation of chemokine receptor Rnd3 30 . Therefore, the PLAM-10 scaffold is expected to recruit stem cells to the bone defect side to promote tissue regeneration in vivo.

In vitro immunoregulation of PLAM-MPN
Immune regulation plays an important role in the osteogenesis process of bone defects, and the polarization and secretion of macrophages are particularly critical 31 . In order to explore the regulatory effect of these scaffolds, BMDMs (95.6% purity, Figure S1) were co-cultured with each sample and their polarization behavior was analyzed by ow cytometry, qRT-PCR and immuno uorescence staining ( Figure   3a). The results of ow cytometry showed that the BMDMs treated with LPS were signi cantly skewed towards the iNOS + M1 subpopulation, while the cells treated with the PLAM-MPN scaffolds signi cantly dampened M1 polarization (Figure 3b, c). The results of qRT-PCR (Figure 3d, e) showed that the PLAM-MPN scaffolds signi cantly down-regulated the expression levels of M1 type-related genes, including iNOS and TNF-α. Meanwhile, there was no statistical difference in the expression of M2 subsets or M2related CD206 and Arg-1 genes among the PLAM-MPN groups ( Figure S2). The immuno uorescence staining (Figure 3f, g) of iNOS in BMDMs further con rmed the immunoregulatory ability of PLAM-MPN, which effectively reduced the in ammatory response by inhibiting the polarization of macrophages towards M1 type and the expression of pro-in ammatory factors, which may be related to the inhibition of macrophage TLR4 signaling pathway by TA in the MPN coating 18 .

In vitro vascularization of endothelial cells on the MPN nanointerface
Angiogenesis is an integral step during regeneration process as the vascular network formation are critical for providing nutrition for the repairing cells. In this context, vascular endothelial cells play a crucial role in maintaining the physiological functions of developing blood vessels 32 . Therefore, HUVECs were used in vitro to characterize angiogenesis inducing ability of MPN nanointerface by tubule formation assay and qRT-PCR analysis (Figure 4a). As shown in Figure 4b-f, the PLAM-10 group exhibited stronger angiogenic capacity, and higher node counts, nodes, grids, and total tube length compared to the control and PLAM groups. In addition, the expressions of angiogenic genes such as HIF, SCF and VEGF [33][34][35] were further assessed by qRT-PCR. Compared with other groups, the relative expression levels of angiogenic genes were signi cantly up-regulated in PLAM-10 group (Figure 4g-i). These results indicated that the MPN nanointerface in PLAM-10 membrane had a strong induction effect on the angiogenesis of HUVECs, which is bene cial for periodontal bone regeneration.

In vitro stem cell osteoinductive ability of the PLAM-MPN
An ideal material for repairing periodontal bone defects should have the ability to enhance the osteogenic differentiation of PDLSCs 36,37 . The mineralized nodule formation ability of PDLSCs was detected by Alizarin red staining and CPC colorimetry after co-culturing these cells with the different scaffolds for 21 d (Figure 5a). Alizarin red staining of the calci ed extracellular matrix clearly demonstrated the enhanced mineralized nodule formation of PDLSCs cultured on the porous side of membrane with MPN coating (Figure 5b). The results of calcium mineral deposition measured by CPC colorimetry further quantitatively con rmed the osteoinductive effect of MPN with nearly 3-fold of increase (Figure 5b).
In addition, the gene expression levels of osteogenic markers, including ALP, Runx2 and OPN, after osteogenic induction for 7, 14 and 21 d were detected by qRT-PCR, respectively. ALP is an early marker of osteoblast differentiation and plays a key role in calci cation in vitro 38 . As shown in Figure 5c, ALP in PLAM-10 group was signi cantly up-regulated by more than 5-fold compared with the NC group at 7 d. Runx2 is an essential transcription factor for osteoblast differentiation, which could promote bone formation and inhibit bone resorption 39 . The expression level of Runx2 in PLAM-10 group was signi cantly increased at 7 d compared with other groups, and continued to increase at 14 and 21 d (Figure 5d). OPN is a matrix protein secreted by osteoblasts and plays an important role in bone regeneration 40 . The expression level of OPN in the PLAM-10 group gradually increased after induction, reaching the maximum level at 21 d, and was signi cantly higher than that in other groups (Figure 5e).
The above results indicated that the MPN coating promoted osteogenic differentiation of PDLSCs.
Meanwhile, the expression of all osteogenic genes was not statistically different between the PLAM scaffold and the NC group, indicating that the pristine PLAM scaffold itself cannot regulate osteogenic differentiation, consistent with the results reported in Figures 5d-f. The mechanism may be that Cu 2+ upregulates the expression of HIF-1α in bone marrow mesenchymal stem cells by activating the Erk1/2 signaling pathway 41,42 , resulting in the secretion of VEGF and BMP-2 proteins, thereby achieving the goal of promoting bone formation.

In vivo bone regeneration of the PLAM-MPN
The above in vitro results revealed the barrier function of the Janus porous structure and the regulation of different bone repairing-related cells based on the MPN nanointerface. This motivated us to evaluate their in vivo biomimetic bone regeneration ability in a rat periodontal bone defect model ( Figure 6a). As shown by the Micro-CT results, the bone defect implanted with the PLAM-10 scaffolds were rich in more newly formed bone tissue compared with the other two groups (Figure 6b). To represent new bone formation in a quantitative and qualitative way, we evaluated various bone mass-related parameters, including BV/TV and BS/TV 24,43 . As could be seen from the Figure 6c, the BV/TV of the PLAM-MPN group was much higher than that of the other two groups at week 1, 2 and 4. Notably, the BV/TV of the PLAM-10 group was still higher than the other two groups at 8 w, which was consistent with the Micro-CT images. The BS/TV values showed a similar trend, that is, the BS/TV values of the PLAM-10 group were signi cantly higher than those of the other two groups at 1, 2, 4, and 8 w after implantation (Figure 6d). In addition, the Tb.Th of the PLAM-10 group was signi cantly higher than that of the other two groups at 2, 4 and 8 w (Figure 6e). Conversely, Tb.Sp decreased over time, indicating that newly formed bone gradually became denser and more mature (Figure 6f). In conclusion, the PLAM-10 scaffolds not only signi cantly improved bone mass (BV/TV and BS/TV), but also signi cantly promoted bone quality (Tb.Th and Tb.Sp) during the entire bone regeneration process. Moreover, there was no signi cant difference in the evaluation of Micro-CT related parameters between the NC group and the PLAM group, indicating that the scaffolds alone were not able to promote bone regeneration, and the modi cation of the MPN active coating greatly improved the osteogenic effects in vivo periodontal bone defects. The results of immuno uorescence staining showed that the expression levels of ALP and Runx2 in bone defects in the PLAM-10 group were signi cantly higher than those in other groups (Figure 6g-j), consistent with the in vitro study that PLAM-10 promoted the osteogenic differentiation of PDLSCs. This study con rmed that PLAM-10 can promote the differentiation of PDLSCs into osteoblasts by increasing the recruitment and attachment of PDLSCs.
Compared with PLAM, PLAM-10 not only enhanced bone mass, but also improves the quality of new bone, and successfully augmented the bone regeneration.

In vivo evaluation of angiogenesis and immune regulation
Bone repair and regeneration are closely related to immune regulation and nutritional support 44 . In order to evaluate the regulatory effect of PLAM-10 on immune cells and angiogenesis in vivo, the related indexes in bone defect side were observed at 2 w after operation. The results of immuno uorescence staining showed that the number of iNOS + cells at the bone defect side in the PLAM-10 group was signi cantly smaller than that in the other groups (Figure 7a, b), while the proportion of CD206 + cells was higher (Figure 7c, d), indicating that PLAM-10 inhibited the polarization of local macrophages towards pro-in ammatory M1 subtype and shifted the polarization towards anti-in ammatory M2, thereby regulating the local in ammatory microenvironment and facilitating tissue regeneration. At the same time, the number of CD31 + cells in the injury center of the PLAM-10 group signi cantly increased ( Figure  7e, f), which indicated that the scaffold material enhanced angiogenesis at the bone detect side.
Moreover, from the SEM imaging of the membranes taken out 8 w after surgery, it was observed that there was almost no in ltration of cells on the barrier surface of PLAM and plenty of cells in ltrated into the porous side ( Figure S3), which also indicated that the Janus porous structure played as a good barrier function in the process of bone repairing. This membrane can block the in ltration of peripheral in ammatory cells and fast-growing soft tissue, which is conducive to regulate and improve the microenvironment of bone defect sides and promote repair.

Conclusion
The effective repair of periodontal bone defects is still a major clinical challenge. At the same time, the development of barrier membrane materials with barrier and bone regeneration-promoting functions is an important research direction towards periodontal tissue regeneration. In this study, a Janus porous PLAM-MPN scaffold was constructed, the dense side acting as a barrier, and the other side being porous as a scaffold for guiding bone regeneration. The MPN bioactive nanointerface was introduced in guided tissue regeneration membrane for the rst time, and it was proved that the MPN nanocoating on PLAM signi cantly inhibited the pro-in ammatory state of macrophages, promoted angiogenesis of HUVECs and enhanced the osteogenesis of PDLSCs. In a rat periodontal bone defect model, the PLAM-MPN barrier membrane facilitated the repair of periodontal bone defects by synergistically regulating the immune, angiogenesis, and osteogenic processes. Therefore, this Janus porous barrier membrane with an effective MPN bioactive nanointerface provides a powerful biomimetic regeneration strategy for realizing periodontal bone defect repair.
On the other hand, the Janus porous membrane based on FDA-approved PLA polymer has a simple preparation method, low cost of raw materials and easy large-scale processing. Meanwhile, the MPN coating demonstrated in this paper that it can be considered a simple and general coating method for various substrates. Considering the wide variety of polyphenols and metal ions and their versatile biological activities, this strategy can be adopted to other barrier membranes as well as to many other forms of tissue engineering materials.

Declarations Author Contributions
Yaping Zhang and Yi Chen equally contributed to conception, design, data analysis, acquisition, and interpretation, drafted and critically revised the manuscript, Tian Ding, Yandi Zhang, Daiwei Yang, Yajun Zhao and Jin Liu contributed to data acquisition, analysis, and interpretation, critically revised the manuscript, Baojin Ma and Alberto Bianco contributed to design, analysis, and interpretation, critically revised the manuscript, Shaohua Ge and Jianhua Li contributed to conception, design, data analysis, and interpretation, critically revised the manuscript. All authors gave nal approval and agree to be accountable for all aspects of the work.  μm. * P <0.05, ** P < 0.01 and *** P < 0.001.     In vivo evaluation of the role of scaffolds in immune regulation and angiogenesis. Representative immuno uorescence staining images and quantitative analysis of bone defects at week 2 post-operation.